FAM69C functions as a kinase for eIF2α and promotes stress granule assembly

Abstract Stress granules are dynamic cytoplasmic ribonucleoprotein granules that assemble in response to cellular stress. Aberrant formation of stress granules has been linked to neurodegenerative diseases. However, the molecular mechanisms underlying the initiation of stress granules remain elusive. Here we report that the brain‐enriched protein kinase FAM69C promotes stress granule assembly through phosphorylation of eukaryotic translation initiation factor 2 (eIF2α). FAM69C physically interacts with eIF2α and functions as a stress‐specific kinase for eIF2α, leading to stress‐induced protein translation arrest and stress granule assembly. Primary microglia derived from Fam69c knockout mice exhibit aberrant stress granule assembly in response to oxidative stress and ATP. Defective stress granule assembly in microglia correlates with the formation of ASC specks and NLRP3 inflammasome activation, whereas induction of stress granule precludes inflammasome formation. Consistently, increased NLRP3 levels, caspase‐1 cleavage and Il18 expression corroborate microglia‐associated neuroinflammation in aged Fam69c knockout mice. Our study demonstrates that FAM69C is critical for stress granule assembly and suggests its role in the regulation of microglia function.


Introduction
Translational control in eukaryotic cells is critical for cell survival during stress responses (Sonenberg & Hinnebusch, 2009). Regulation of protein translation is imposed at the initiation stage. The phosphorylation of the a-subunit of eukaryotic translation initiation factor 2 (eIF2a) decides the integrity of the preinitiation complex and translation initiation . There are four known kinases that are responsible for the phosphorylation of eIF2a under specific conditions, including PKR-like ER kinase (PERK), doublestranded RNA-dependent protein kinase (PKR), heme-regulated eIF2a kinase (HRI), and general control non-derepressible protein 2 (GCN2) (Pakos-Zebrucka et al, 2016).
Stress granules (SGs) form when translation initiation is inhibited during a variety of stress responses, including heat shock (HS), nutrient deprivation, hypo-osmotic conditions, and oxidative stress (Protter & Parker, 2016). SGs have been proposed to stabilize mRNAs, regulate mRNA translation and have impact on physiological and pathological processes (Buchan & Parker, 2009). SG assembly entraps viral mRNAs and inhibits viral gene expression in antiviral immune response (McCormick & Khaperskyy, 2017). Phosphorylation of eIF2a and SG assembly promote the survival of cells under stress (Aulas et al, 2017). In tumor cells, SG assembly inhibits mTORC1 and apoptosis through SG component astrin (Thedieck et al, 2013). SG assembly protects neuron and other long-lived cells from death under stress (Farley & Watkins, 2018). Mutation of FUS (Baron et al, 2013;Bosco et al, 2010) and expanded C9ORF72 hexanucleotide repeat (Boeynaems et al, 2017) in neurodegenerative diseases are associated with defects in the disassembly of SGs, resulting in the phosphorylation of cytosolic TDP43 and inhibition of nuclear import (Gasset-Rosa et al, 2019;Wolozin & Ivanov, 2019).
Neurodegeneration is characterized by progressive loss of neuron activity and function, ultimately leading to neuron death. It is well known that pathologic protein aggregation triggers neurodegeneration in neurodegenerative disease. Amyloidosis, tauopathies, asynucleinopathies, and TDP-43 proteinopathies represent the predominant type of pathological hallmarks (Dugger & Dickson, 2017;Vaquer-Alicea & Diamond, 2019). Pathologic protein aggregates have been considered to be the cause of neurodegeneration, while the immune changes in neurodegenerative diseases were considered as a secondary or reactive response to protein aggregates and neurodegeneration. With the development of sequencing technology, the microglial-specific gene TREM2 R47H mutation identified with genome-wide association studies (GWASs) suggest that dysregulated immune responses contribute to the disease etiology (Jonsson et al, 2013).
Microglia are the main resident innate immune cells in the central nervous system (CNS). Like macrophages, microglia activation can be categorized as either classical (M1) or alternative (M2), representing the proinflammatory and anti-inflammatory states of microglia (Colonna & Butovsky, 2017). Microglia not only recognize pathogen associated molecular patterns (PAMPs) but also can be activated by danger-associated molecular patterns (DAMPs) or protein aggregates (Labzin et al, 2018). Extracellular misfolded and aggregated proteins can activate microglia through pattern recognition receptors. Activation of Toll-like receptors (TLRs) triggers NF-jB-dependent proinflammatory gene expression and the induction of proinflammatory cytokines (Lehnardt, 2010). Moreover, microglia express various purinergic receptors for ATP, such as P2Y12 and P2X4, to detect cell injuries (Colonna & Butovsky, 2017). NLRP3 inflammasome mediates M1 macrophage polarization (Zhang et al, 2020) and has been found in various neurodegenerative diseases including Alzheimer's disease (Heneka et al, 2015), frontotemporal dementia (McCauley & Baloh, 2019), and Parkinson's disease (Marogianni et al, 2020). NLRP3 inflammasomes induce the cleavage of caspase-1, which is responsible for the maturation and secretion of the inflammatory cytokines Interleukin-1b (IL-1b) and IL-18, and additional pyroptosis (Kanneganti et al, 2006;Voet et al, 2019). Moreover, previous studies implicated that NLRP3 inflammasomes are essential for the development and progression of amyloid-b (Ab) pathology and tau pathology in mouse models (Venegas et al, 2017;Ising et al, 2019).
As the predominant types of stress responses, crosstalk between stress granule and NLRP3 inflammasome has been studied. On one hand, inflammation upregulated eIF2a phosphorylation and stress granule formation in heat-shocked intestinal epithelial cells (Hu et al, 2010), whereas anti-inflammatory cytokine treatment reversed eIF2a phosphorylation and SGs formation (Herman et al, 2019). On the other hand, SG assembly inhibits the formation of NLRP3 inflammasomes through the sequestration of HSP90 and DDX3X in SGs (Mayor et al, 2007;Samir et al, 2019). Additionally, SG assembly directly affects phagocytosis of microglia through the sequestration of SYK (Ghosh & Geahlen, 2015).
In our previous studies, we demonstrated that FAM69C is a brain-enriched kinase that regulates synaptic plasticity and memory and is associated with neurodegenerative dementia (Mei et al, 2022). In this study, we explore the function of FAM69C as an eIF2a kinase that promotes stress granule assembly and suggest its role in the regulation of microglia function.

FAM69C is a stress-specific kinase for eIF2a
In our previous work, we characterized a brain-enriched kinase FAM69C and demonstrated that FAM69C deficiency is associated with neurodegenerative dementia. To unveil the biological function of FAM69C, we performed S-tag pulldown assay to identify its interactive proteins. Mass spectrometry analyses identified a total of 1,355 potential candidates associated with FAM69C protein (as assessed by > 5-fold intensity as compared to HA-S-tag control, Dataset EV1). Biological pathway analyses indicated that these candidates were enriched in pathways relative to stress granule (SG) assembly, regulation of translation in response to stress, ribonucleoprotein complex assembly, and cytoplasmic translation ( Fig 1A). These results indicate that FAM69C is potentially involved in stress responses. Notably, EIF2S1-encoded eIF2a protein is a key component of the preinitiation complex that initiates protein translation (Wolozin & Ivanov, 2019), and the reversible phosphorylation of eIF2a mediates stress-induced protein synthesis arrest and SG assembly (McCormick & Khaperskyy, 2017). Thus, we speculated that eIF2a was a critical molecule that coupled regulation of translation in response to stress and SG assembly, the two FAM69C-associated biological pathways. Next, we examined the physical interaction of FAM69C and eIF2a. Under the basal condition, immunoprecipitation in SH-SY5Y cells indicated that the endogenous eIF2a interacted with FAM69C. Further, we treated SH-SY5Y cells with SG inducer sodium arsenite (AS) and observed that the interaction of FAM69C and eIF2a was significantly strengthened (Fig 1B and C).
Based on the evident interaction between eIF2a and FAM69C, we next determined whether eIF2a might be a substrate of FAM69C. We treated FAM69C +/+ and FAM69C À/À SH-SY5Y cells with AS or heat shock (HS), which were the two conditions that strictly depend on eIF2a for SG induction. Notably, western blotting revealed that levels of phosphorylated eIF2a at S51 in FAM69C +/+ cells were significantly increased upon AS or HS treatment, whereas loss of FAM69C deteriorated eIF2a phosphorylation (Fig 1D and E). By contrast, the D279N mutant, in which the kinase activity was impaired by substituting the critical amino acid Asp 279 in the catalytic loop with Asn, reduced eIF2a phosphorylation ( Fig EV1A). Further, we determined whether FAM69C directly phosphorylates eIF2a using recombinant FAM69C 44-419 and full-length eIF2a proteins. In the in vitro phosphorylation assay, FAM69C protein directly phosphorylated eIF2a, as indicated by western blotting (Fig 1F). Consistently, the ADP-Glo kinase assay corroborated that FAM69C phosphorylates eIF2a through its kinase activity (Figs 1G and EV1B). These data indicated that FAM69C functions as a kinase for eIF2a.
Given that four known kinases phosphorylate eIF2a under specific condition, we determined whether FAM69C is a stress-specific kinase for eIF2a. We examined three conditions that relate to SG assembly, including oxidative stress, heat shock, and ER stress. First, we knocked down HRI in the FAM69C +/+ and FAM69C À/À SH-SY5Y cells and tested the levels of phosphorylated eIF2a in response to oxidative stress (Fig EV1C and D). Western blotting showed that knockout of FAM69C substantially reduced phosphorylated eIF2a upon AS treatment ( Fig EV1C, lane 7 versus lane 8). Further reduction in HRI abolished eIf2a phosphorylation ( Fig EV1C, lanes 11, 12 versus lanes 9, 10). These data indicated that both FAM69C and HRI are the physiologically relevant kinases that phosphorylate eIF2a under AS-induced oxidative stress. Second, we knocked down PERK in the FAM69C +/+ and FAM69C À/À cells. Upon heat shock, levels of phosphorylated eIF2a were significantly increased in FAM69C +/+ cells, as compared with that in FAM69C À/À cells (Fig EV1E, lane 7 versus lane 8). Further, knock down of PERK did not impose much effect on the levels of phosphorylated eIF2a ( Fig EV1E, lanes 9, 10 versus lanes 11, 12). These data revealed that FAM69C, but not PERK is responsible for phosphorylating eIF2a upon heat shock. Third, we determined the kinase for eIF2a under ER stress. In thapsigargin (TG) treatment, levels of phosphorylated eIF2a were considerably increased, which was independent of FAM69C ( Fig EV1E,  lane 13 versus lane 14). However, knock down of PERK completely inhibited eIF2a phosphorylation (Fig EV1E,lanes 15,16,17,18 versus lanes 13,14). These data indicated that PERK, but not FAM69C is the responsible kinase for eIF2a under ER stress. Taken together, we propose that FAM69C is a stress-specific kinase that phosphorylates eIF2a under oxidative stress and heat shock, but not ER stress.

FAM69C mediates stress-induced translation arrest and promotes stress granule assembly
Given that FAM69C is a kinase of eIF2a and phosphorylated eIF2a rapidly arrests protein synthesis, we thus examined the role of FAM69C in stress-induced translation inhibition by RiboPuromycylation assay. In FAM69C +/+ SH-SY5Y cells, western blotting revealed that protein translation was severely inhibited in response to AS treatment ( Fig 2B). These data indicated that FAM69C deficiency led to aberrant stress-induced translation inhibition. Based on the fact that SG assembly parallels stress-induced translation inhibition, we examined the role of FAM69C on SG formation. We constructed an SH-SY5Y cell line with stably expressed GFPtagged G3BP1 for live-cell imaging. FAM69C +/+ and FAM69C À/À SH-SY5Y cells were treated with AS to induce SGs, and the formation of SGs was continuously monitored for 45 min. Interestingly, FAM69C deficiency resulted in a significant delay in the rate of SG assembly, as compared to that of FAM69C +/+ cells (Fig 2C and D). Immunofluorescence indicated that FAM69C did not colocalize with G3BP1 ( Fig EV2A), suggesting that FAM69C was not a component of SG. These data indicated that FAM69C promotes SG assembly through upstream eIF2a signaling.

FAM69C facilitates stress granule assembly in microglia
Microglia, as the primary immune cell in the brain, actively respond to stress conditions and are linked to a variety of neurodegenerative diseases. We thus determined the role of FAM69C in the regulation of stress granule in microglia. Primary microglia derived from wildtype and Fam69c À/À mice were treated with a series of conditions ( Fig EV3A), and SGs were labeled with endogenous G3BP1. Under the basal condition, there were no SGs in either genotype ( Fig 3A).
Upon AS treatment, Fam69c +/+ microglia form a greater number of SGs, as compared to that in the Fam69c À/À microglia ( Fig 3B and D). In addition to oxidative stress, microglia sense damage-associated ATP through purinoceptors, a process that controls microglial chemotaxis to injury (Li & Barres, 2018). Remarkably, exposure to ATP induces robust SGs formation in Fam69c +/+ microglia, whereas less SG assembly was observed in Fam69c À/À microglia (Fig 3C and D). Taken together, we suggest that both AS and ATP induce FAM69Cdependent SGs formation in microglia.

FAM69C-dependent stress granule assembly in microglia is correlated with reduced NLRP3 inflammasome activation
Given that reversible SG assembly plays a protective role for eukaryotes cells to survive stresses, we examined the cellular effect of SG assembly in microglia. Microglia, a type of brain-resident macrophage, is activated by misfolded proteins, oxidative stress, and ATP in neurodegenerative diseases and exerts multifaceted functions (Song & Colonna, 2018). The cytosolic innate immune signaling receptor NLRP3 senses these damage-associated molecular patterns (DAMPs) and mediates inflammasome activation . Specifically, NLRP3 inflammasome activation involves a priming signal that results in the NF-jB-dependent transcriptional upregulation of NLRP3 and pro-IL-1b/18, and the following activation signal that induces the oligomerization and activation of NLRP3 (Voet et al, 2019). We thus primed microglia with lipopolysaccharide (LPS) and observed no difference in the priming step of NLRP3 activation between Fam69c +/+ and Fam69c À/À microglia (Fig EV3B and C). Meanwhile, LPS treatment did not affect SG assembly ( Fig 4A). Next, we induced SG formation in microglia by adding ATP after 3 h of LPS priming. A greater number of SGs were found in Fam69c +/+ microglia, compared with that in Fam69c À/À microglia. Interestingly, we observed significantly fewer ASC specks in Fam69c +/+ microglia than that in Fam69c À/À microglia (Fig 4B and  C). These data indicated that FAM69C-dependent SG formation is correlated with reduced NLRP3 inflammasome activation.
Further, we explored the potential mechanism underlying the negative correlation of SG assembly and NLRP3 inflammasome. First, we determined whether FAM69C was a component of inflammasome. Immunofluorescence showed that FAM69C was not colocalized with NLRP3 ( Fig EV3D). As previously reported that sequestration of ◀ Figure 1. FAM69C is a stress-specific kinase for eIF2a.
A Cnetplot of enriched pathways for 'cytoplasmic translation', 'regulation of translation in response to stress', 'ribonucleoprotein complex assembly' and 'stress granule assembly'. S-tag pull-down of FAM69C in SH-SY5Y cells and consequent mass spectrometry identified a series of potential proteins interacting with FAM69C. B Immunoblot analysis of immunoprecipitates with endogenous eIF2a and HA-S-tagged FAM69C. SH-SY5Y cells were transfected to express HA-S-FAM69C, followed by 0.5 mM AS treatment for 30 min. HA-S-FAM69C was pulled-downed with S-protein beads, and the S-protein immunoprecipitants were immunoblotted for eIF2a and HA. Representative blots, n = 3 biologically independent experiments. C Quantitative analysis of eIF2a in the FAM69C Pull-down assay. *P value < 0.05, paired two-tailed t-test, n = 3 biologically independent experiments. Mean AE SD. For FAM69C versus mock, P value = 0.0103; For FAM69C versus FAM69C-AS, P value = 0.0289. D Immunoblot analysis of phosphorylated eIF2a in FAM69C +/+ and FAM69C À/À SH-SY5Y cells. Cells were treated with 0.5 mM AS, 42°C heat shock for 30 min, and lysates were immunoblotted with p-eIF2a (S51) antibody. Representative blots, n = 3 biologically independent experiments. E Quantitative analysis of the phosphorylation level of eIF2a normalized with total eIF2a. *P value < 0.05, paired two-tailed t-test, n = 3 biologically independent experiments. Mean AE SD. For AS, P value = 0.0196; For HS, P value = 0.0335. F Immunoblot analysis of phosphorylated eIF2a in the in vitro phosphorylation assay. 10 lg recombinant eIF2a protein was mixed with or without 1 lg FAM69C protein in reaction buffer (40 mM Tris-HCl (pH7.5), 20 mM MgCl 2 , 1 mM ATP) at 37°C for 30 min. Phosphorylated eIF2a was detected with the p-eIF2a (S51) antibody. Representative blots, n = 3 biologically independent experiments. G The relative luminescence in ADP-Glo kinase assay. ADP Generated by FAM69C and the kinase-dead mutant D279N in the in vitro phosphorylation assay was quantified with ADP-Glo kinase assay, BSA was used as a control. **P value = 0.0084, *P value = 0.0239, paired two-tailed t-test, n = 4 biologically independent experiments. Mean AE SD. DDX3X in stress granule prevents its interaction with NLRP3 and inflammasome activation (Samir et al, 2019), we determined the status of DDX3X sequestration in Fam69c +/+ and Fam69c À/À microglia.
As expected, ATP exposure induces SG assembly and DDX3X sequestration in SG in Fam69c +/+ microglia, as compared with control ( Fig 4D). By contrast, loss of FAM69C failed to sequester DDX3X.
A Immunoblot analysis of de novo synthesized proteins in SH-SY5Y cells. Puromycin incorporation assay of FAM69C +/+ and 2 FAM69C À/À SH-SY5Y cell lines treated with 0.5 mM AS for 30 min followed by 50 lg/ml puromycin for 15 min. An anti-puromycin antibody (Puro) was used to visualize de novo synthesized proteins. A representative image is shown, n = 3 biologically independent experiments. B Quantitative analysis of puromycin labeled de novo synthesized proteins. *P value < 0.05, paired two-tailed t-test, n = 3 biologically independent experiments.

FAM69C-independent inflammasome activation
It has been well characterized that NLRP3 inflammasome is induced by various inducers, through potassium-dependent and potassiumindependent mechanisms (Mangan et al, 2018). Based on the findings that FAM69C regulates ATP-induced SGs in microglia and correlated with reduced inflammasome activation, we examined the role of FAM69C in other induction conditions. Nigericin is one of the most commonly used inflammasome inducers, which permeabilizes the cell membrane specifically to potassium. Without priming, Fam69c +/+ and Fam69c À/À microglia did not show inflammasome activation upon nigericin treatment (Fig 5A). After LPS priming, nigericin induced a comparable number of ASC specks in Fam69c +/+ and Fam69c À/À microglia, in the absence of SG formation (Fig 5B and C).
To corroborate these findings, we employed the lysosomotropic reagent L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) to disrupt lysosomes. Likewise, Leu-Leu-OMe did not induce SGs, but activated comparable number of NLRP3 inflammasome independent of FAM69C genotype both in the primed and unprimed microglia (Fig 5D-F). Taken together, we proposed that FAM69C-associated inflammasome inhibition in microglia is dependent on conditions that induce SG assembly. To this end, ATP exposure, but not other inflammasome inducers, is related to the crosstalk between FAM69Cdependent SG assembly and inflammasome activation in microglia.

SG assembly precludes NLRP3 inflammasome activation
To examine whether the correlation between SG assembly and inflammasome inhibition is a general observation, we induced SG assembly in microglia and followed by treatment with the inflammasome inducer. To this end, microglia were primed with LPS, pretreated with AS to induce SG assembly, and followed by nigericin treatment (Fig 6A). Remarkably, SG assembly did preclude NLRP3 inflammasome activation (Fig 6B). Sorbitol, like AS, was able to induce SG assembly in primed microglia ( Fig EV3E). Consistently, we found that the presence of sorbitol-induced SGs inhibited nigericin-induced inflammasome formation (Fig 6C and D). Next, we tried to rescue exacerbated inflammasome activation in Fam69c À/À microglia. We prolonged the AS treatment from 20 to 40 min and followed by ATP treatment. Prolonged AS treatment was sufficient for the finalization of delayed SG assembly in Fam69c À/À microglia (Fig 6E). With the presence of SGs, inflammasome activation represented by ASC specks was completely suppressed in Fam69c À/À microglia (Fig 6F). Taken together, we proposed that SG assembly precludes NLRP3 inflammasome formation.

Aged FAM69C-deficient mice show inflammasome activation
As the predominant type of innate immune cells in the brain, microglia are the main source of proinflammatory cytokines. Cytokines released by microglia are key mediators of neuroinflammation, which can induce and regulate a wide range of cellular responses (Colonna & Butovsky, 2017). The homeostasis of microglia is thus critical for normal brain function. Moreover, proinflammatory cytokines lead to dysfunctional microglia and dampen the clearance of misfolded proteins in neurodegenerative disease.
Based on these findings in primary microglia derived from Fam69c +/+ and Fam69c À/À mice, we determined the effect of FAM69C in microglia-mediated inflammation in vivo. Given that aging parallels the induction of inflammation, we classified mice into 3-month-old young group and 12-month-old aged group. Cortical tissues from the aged Fam69c À/À mice showed significantly increased NLRP3 levels and caspase-1 cleavage, indicating inflammasome activation (Fig 7A and B). Moreover, we examined caspase-1-mediated proteolytic activation of cytokines. qRT-PCR demonstrated that the expression of Il18 and its receptor Il18r1 were significantly upregulated in the aged Fam69c À/À mice ( Fig 7C). Further, immunofluorescence revealed an increased number of ASCexpression microglia in the medial prefrontal cortex (mPFC) of aged Fam69c À/À mice than that from Fam69c +/+ mice (Fig 7D and E). Consistently, we observed a greater number of eosinophilic neurons in the cortex of aged Fam69c À/À mice, as compared with the Fam69c +/+ mice (Fig 7F). By contrast, no inflammasome activation was observed in the 3-month-old young Fam69c À/À mice ( Fig EV4A). HE staining showed no evident difference between Fam69c À/À mice and age-matched young Fam69c +/+ mice ( Fig EV4B). These data indicated that FAM69C deficiency together with aging parallels microglia-associated inflammasome activation.
Taken together, these data suggested that aged FAM69Cdeficient mice show inflammasome activation.

Discussion
In our previous study, we identified that FAM69C is a brainenriched kinase linked to neurodegenerative diseases (Mei ◀ Figure 4. Stress granule assembly in microglia is correlated with reduced NLRP3 inflammasome activation. A, B Confocal imaging of LPS (1 lg/ml, 3 h) primed primary cultured Fam69c +/+ and Fam69c À/À microglia treated with or without 5 mM ATP for 40 min to visualize the formation of stress granules (green, indicated by arrowheads) and inflammasomes (red, indicated by arrowheads). LPS-primed Fam69c À/À microglia showed impaired ATP-induced stress granule assembly compared with Fam69c +/+ microglia when treated with ATP. Meanwhile, more cells with ASC specks were found in Fam69c À/À microglia than that in Fam69c +/+ microglia. Representative images, n > 3 biologically independent experiments. Scale bar = 10 lm. C Quantitative analysis of microglia with stress granules and inflammasomes treated with LPS, LPS and ATP. ****P value < 0.0001, unpaired two-tailed t-test. For LPS, n = 15 fields from three biologically independent experiments; For LPS + ATP, n = 40 fields from six biologically independent experiments. Mean AE SD. D Confocal imaging of primary cultured Fam69c +/+ and Fam69c À/À microglia treated with mock, 5 mM ATP (40 min), 1 lg/ml LPS (3 h) and 5 mM ATP (40 min) to visualize DDX3X (red, indicated by arrowhead) localization. G3BP1 was used as a marker of stress granules. Fam69c +/+ microglia form stress granules and lead to the sequestration of DDX3X when treated with ATP. Scale bar = 10 lm.
et al, 2022). The substrates of FAM69C and its biological function are largely unknown. Here, we found that FAM69C functions as an eIF2a kinase and promotes stress granule assembly. Specifically, we characterized the impact of FAM69C-dependent stress granule assembly on microglia function. This study suggests the potential protective role of FAM69C in aging and neurodegenerative diseases. The cellular stress response has a vital role in the regulation of brain homeostasis (Farley & Watkins, 2018). eIF2a is a critical molecule in stress responses, related to integrated stress response and stress granule assembly. Phosphorylation of eIF2a suppresses general protein translation and subsequently promotes SG assembly, which is composed of condensates of RNA-binding proteins and untranslated messenger RNAs. Here, we found that ATP is able to induce SG assembly in microglia. We provided evidence that ATP treatment leads to increased levels of phosphorylated eIF2a. By contrast, the inflammasome inducer nigericin was not able to induce phosphorylation of eIF2a (Fig EV5A and B). Previous studies have employed both ATP and nigericin as inflammasome inducers in macrophages (Iyer et al, 2009;Samir et al, 2019). Here, we showed the difference between ATP and nigericin in the activation of eIF2a signaling and induction of SG assembly in microglia. Further, we provided evidence that FAM69C functions as a novel stress-specific kinase for eIF2a, in addition to the four known eIF2a kinases. We delineate the role of FAM69C in the regulation of SG assembly through eIF2a signaling.
Ó 2023 The Authors EMBO reports 24: e55641 | 2023 microglia, which leads to the sequestration of DDX3X in SG. Meanwhile, we observed decreased inflammasome activation in Fam69c +/+ microglia as compared with Fam69c À/À microglia. These data suggest that FAM69C may indirectly regulate inflammasome activation through DDX3X. Further, we provided evidence that induction of SG assembly in Fam69c À/À microglia rescued exacerbated inflammasome activation. Our work illustrates that FAM69C promotes SG assembly in microglia and regulates microglia function, albeit its impact on reduced inflammasome activation is not direct. Activation of microglia contributes to neuroinflammation (Hammond et al, 2019). Pathological misfolded protein aggregates and DAMPs released from dead neurons can activate microglia through inflammasomes. Innate immunity contributes to the development of neurodegenerative diseases. Inflammasome activation in Fam69c À/À mice was manifested by increased NLRP3 expression, caspase-1 cleavage, and Il18 expression. Changes of another key interleukin IL-1b involved in inflammasome activation were not seen in qRT-PCR, which may be due to the relatively lower expression of Il1b than Il18. In the future study, we may isolate microglia, rather than the whole tissue lysates from the aged Fam69c À/À brain, and measure the expression of Il1b .
In conclusion, we discovered that the brain-enriched kinase FAM69C promotes SG assembly through the phosphorylation of eIF2a. Stress-induced stress granule assembly in microglia precludes NLRP3 inflammasome activation. These findings reveal a new molecular mechanism for the initiation of stress granules and demonstrate the role of FAM69C in the regulation of microglia function.

Animals and cell lines
All animals were maintained in a special pathogen-free facility, and the animal study protocols used were approved by the ethics committee of Peking University Health Science Center (approval number LA2017142). The 293T, Hela, and SH-SY5Y cell lines were obtained from the American Type Culture Collection. The BV2 microglial cell line was purchased from Procell Co., Ltd. and has been authenticated recently (#CL-0493, Wuhan, China).
Fam69c À/À mice and FAM69C À/À cell line Fam69c À/À mice and FAM69C À/À SH-SY5Y cell line used in this study were generated using the CRISPR design tool, which has been described previously by Mei et al (2022).

Antibodies
The following antibodies were used in this study: ASC (

HE staining and ASC staining
Mice were sacrificed for HE staining and ASC staining. The paraffinembedded brain was sectioned into 5 lm slices, followed by HE staining. For ASC staining, frozen sections were used. The sections were blocked in 5% BSA, 0.3% Triton X-100 and PBS (blocking buffer) for 30 min followed by 3 h of room temperature incubation with the ASC antibody (#67824, CST) in the blocking buffer. Sections were washed three times in PBS and incubated with Alexa Fluor 555 conjugated secondary antibodies for 60 min, followed by DAPI for 10 min. To quench autofluorescence, the sections were incubated for 20 min in 0.1% Sudan Black B in 75% ethanol and washed extensively in PBS.
Proteins were separated by SDS-PAGE and blotted on PVDF membranes. The membranes were blocked with 5% nonfat milk in PBS-T (1% Tween 20)/TBS-T (20 mM Tris, Ph8.0, 150 mM NaCl with 0.5% Tween 20) for 1 h at RT and incubated with the indicated antibodies overnight at 4°C. After washing with PBS-T/TBS-T buffer three times, membranes were incubated with HRP-conjugated secondary antibodies for 1 h at RT.

Immunofluorescence
Cells seeded on glass coverslips were fixed with 4% PFA at room temperature for 15 min, followed by blocking in 1% bovine serum albumin containing 0.3% tritonX-100 at room temperature for 1 h, and incubation in the diluted primary antibody at 4°C overnight. After being incubated with secondary antibodies at room temperature for 30 min and stained with 0.5 lg/ml DAPI for 10 min, coverslips were mounted with fluorescence decay resistant medium. Images were acquired with a Nikon TCS A1 microscope.
FAM69C was obtained as described by Mei (Mei et al, 2022). For recombinant expression of eIF2a, DNA fragments encoding human eIF2a were cloned into a pET-28a (+) vector with His tag on the N terminal. BL21 cells transfected with eIF2a were treated with 0.1 mM IPTG for 20 h and then lysed by ultrasonic. eIF2a was enriched with Ni Beads.

Live-cell imaging
Cells stably expressing GFP-tagged G3BP1 were established for livecell imaging. FAM69C +/+ or FAM69C À/À cells were seeded on cell culture dishes (Bioptechs Inc) 24 h before imaging. The heater was used to keep the temperature at 37°C. Multipoint images were taken every 90 s with the 488 nm laser after 0.1 mM AS treatment immediately.

Membrane and cytoplasm isolation
HEK 293T Cells expressing C-terminal GFP tagged FAM69C were grown to confluency in a 150 mm dish and treated with or without 0.5 mM sodium arsenite (AS) for 1 h. Membrane and cytoplasm were isolated with Applygene P1201. For density gradient centrifugation, cells were lysed in CER buffer (Applygene P1201) for 15 s. The lysate was then centrifuged for 5 min at 800 g. 1 ml Supernatant was layered on a linear sucrose gradient (15-45%, 400 ll/ layer, 11 layer), centrifuged at 100,040 g in a Beckman sw55Ti rotor at 4°C for 40 min. Fractions 1-10 (500 ll each) were harvested from the top to the bottom. 15 ll from each fraction was analyzed by western blot. Fraction 1 represents the lowest density fraction, and fraction 10 represents the highest density.

Primary microglia culture
Newborn mice were sacrificed for mixed glial culture, and primary glial cells were further cultured in DMEM supplemented with 10% FCS, 100 U/ml penicillin/streptomycin, and 20 ng/ml GM-CSF. Microglial cells were used after 14 days of primary cultivation. To assess the formation of ASC specks, LPS-primed microglia were activated with nigericin (20 lM) or ATP (5 mM) for 40 min. Cells were then fixed for further immunofluorescence assays.
qRT-PCR mRNA from mice brains was reverse transcribed into cDNA with All-In-One RT MasterMix (Abm), and quantitative real-time polymerase chain reaction (qPCR) was carried out on a 7,500 Fast realtime PCR system.
S-tag pull-down assay SH-SY5Y cells stably expressing S-HA-tagged FAM69C were grown to confluency in a 150 mm dish for S-tag pull-down. Cells were lysed for 30 min in 0.5% NP40 lysis buffer, and FAM69C was recognized and pulled down by the S-protein Agarose beads. The FAM69C-bounded S-protein Agarose beads were washed 3 times with 0.3% NP40 buffer, followed by a mass spectrometry assay. ClueGO-v2.5.8 (Bindea et al, 2009) was used for GO enrichment analysis of potential interacting proteins.

Data availability
The data set (and computer code) produced in this study is available in the following database: Mass spectrometry data: Proteomics Identification Database PXD037697 (https://www.ebi.ac.uk/pride/ archive/projects/PXD037697). Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request. Figure EV1. FAM69C is a stress-specific kinase for eIF2a.
Confocal imaging of SH-SY5Y cells transfected with FAM69C and G3BP1, to visualize the localization of FAM69C and stress granules. Scale bar = 10 lm.
▸ Figure EV3. Features in primary culture studies.
A Confocal imaging of isolated primary microglia. GFAP (Purple) was used as a marker of astrocyte, and Iba1 (Green) was used as a marker of microglia. All of the Iba1-positive microglia are ASC-positive (Red). Scale bar = 10 lm. Representative images, n = 8 fields from three biologically independent experiments. B Immunoblot analysis of protein levels of core NLRP3 inflammasome components in primary cultured Fam69c +/+ and Fam69c À/À microglia after LPS priming (1 lg/ml LPS for 3 h). Representative blots, n = 3 biologically independent experiments. C Quantitative analysis of protein level of NLRP3, ASC and CASP1 normalized with b-Actin in Fam69c +/+ and Fam69c À/À microglia. n = 3 biologically independent experiments. Mean AE SD. D Confocal microscopy imaging of SH-SY5Y cells transfected with FAM69C and NLRP3, to visualize the localization of FAM69C and NLRP3 specks. Scale bar = 10 lm. E Confocal imaging of primary cultured microglia treated with 1 lg/ml LPS (3 h), followed by 0.4 M sorbitol (1 h), to visualize the formation of stress granules.
A Immunoblot analysis of NLRP3 and cleaved caspase1 (p20) in cortex of 3-month-old Fam69c +/+ and Fam69c À/À mice. Data are from four different mice with each genotype. B Hematoxylin and eosin staining of 3-month-old Fam69c +/+ and Fam69c À/À mouse brain. The image is representative of three pairs of mice. Scale bar = 25 lm. Figure EV5. ATP-induced phosphorylation of eIF2a in BV2 cells.
A Immunoblot analysis of phosphorylated eIF2a in BV2 cells. Primed (1 lg/ml LPS for 3 h) or unprimed BV2 cells were treated with mock, 5 mM ATP or 20 lM nigericin for 40 min, and lysates were immunoblotted with p-eIF2a (S51) antibody. Representative blots, n = 3 biologically independent experiments. B Quantitative analysis of the phosphorylation level of eIF2a normalized with total eIF2a. **P value < 0.01, paired two-tailed t-test, n = 3 biologically independent experiments. Mean AE SD. For ATP versus mock, P value = 0.0047; For LPS + ATP versus LPS, P value = 0.0011.